Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

Abstract

We describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [3H]fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [3H]fMet-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, we also demonstrated fMet-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils.

Differential Mechanisms of Recognition and Activation of Interleukin-8 Receptor Subtypes

“…21 are adjacent or are in the surface-exposed hydrophobic pocket that has been argued to drive the association of IL-8 to the receptor by hydrophobic interactions (17). In our present work, substitution of His 18 and Phe 21 for Ala caused modest changes in binding affinity, calcium responses, and release of ␤-glucuronidase. These observations indicate that the hydrophobic nature of these residues does not play a major role in the mechanisms of recognition and activation of the IL-8 receptors.…”

“…Red blood cells were stored in Alsevers solution consisting of 114 mM dextrose, 27 mM sodium citrate, 71 mM NaCl (pH 6.1) at 4°C. Rabbit neutrophil membranes were prepared as described (18).…”

“…Although, these single Ala mutants are poor activators of superoxide production, suggesting that these mutants are not full agonists in terms of production of superoxide. Single or double substitutions of His 18 and Phe 21 by polar or charged residues produce major changes in binding affinity to CXCR1 and the Duffy antigen. In contrast, minor changes were observed with CXCR2.…”

“…These residues are in or adjacent to the surface-exposed hydrophobic pocket in IL-8 which has been previously implicated in the recognition mechanisms to the IL-8 receptors (16,17 (26), suggesting that these residues bind to the N terminus domain of the IL-8 receptors. Single and double IL-8 mutants were created by substitution of His 18 and Phe 21 by Ala, Ser, or Asp. All these mutants exhibited reduced binding to the anti-IL-8 mAb (20).…”

“…1 shows the purity of the IL-8 mutants, H18D and H18A/F21A, as determined by SDS-polyacrylamide gel electrophoresis. To examine whether His 18 and Phe 21 residues play a role in the mechanisms of recognition and activation of the IL-8 receptors, we initially performed displacement of 125 I-IL-8 bound to neutrophil membranes by IL-8 mutants. In Table I we showed that H18A and F21A mutants exhibited 4-and 3-fold increases in the K i , respectively, as compared with that of the wild type IL-8, suggesting a moderate reduction in binding affinity of the mutants toward IL-8 receptors.…”